|Public on May 01, 2016
|sh52 hairpin DOX biological replicate 1
|in vitro transformed human skin fibroblasts
|cell line: CSC_shH1.0-1
treatment: DOX (14d Dox)
|Cells were treated with 1µg/ml Doxycicline (dox) for 16h or 14d to induce knock-down, or let re-express the knocked-down protein by removing dox for 4d after the 14d dox treatment.
|Cells were grown in minimum essential medium (MEM) containing Tet-free 15% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, at 37 °C in 5% CO2. Inducible cell lines were generated by introducing inducible pTRIPZ constructs V2THS_38052 (shH1.0-1) and V2THS_38055 (shH1.0-2) (Open Biosystems) in the cells.
|Total cell RNA was prepared from the cells using RNAeasy reagents (Qiagen).
The Total RNA samples were quality controlled (QC) by using the 6000 Nano RNA Chip on the BioAnalyser 2100 (Agilent, Santa Clara, CA, USA) to insure RNA integrity and concentration before starting the procedure. The Total RNA samples were subjected to Ribozero Gold (Epicentre Madison, U.S.A.) treatment to remove the rRNA fraction from the samples. After this treatment we QC the samples again using the 6000 mRNA RNA Chip on the BioAnalyser 2100 (Agilent, Santa Clara, CA, USA) to check that there was sufficient reduction in the rRNA. If the reduction in the rRNA was sufficient we continue to library preparation (TruSeq Stranded Total RNA-Seq Library Prep 15031048-E. Illumina San Diego, CA, USA). This protocol by Illumina was adjusted to fit the samples sets. We optimised this protocol to make better use of samples of lower RNA integrity and concentration. The standard PCR cycling was also changed to match the concentration of the total RNA from the mRNA QC We also found that the final elution of 32.5µl with resuspension buffer was over diluting the libraries so we eluted using 16µl. After this clean up stage the final QC on the DNA 1000 BioAnalyser 2100 chip (Agilent, Santa Clara, CA, USA) was performed. After passing the final QC the stranded total RNA-seq libraries were ready for Flowcell cluster formation on a cBot and then 101bp Paired End Sequencing by synthesis on the HiSeq 2500 was performed.
|Illumina HiSeq 2500
|Alignments were performed to human RefSeq genes archived in the Illumina iGenomes resource using RSEM (version 1.2.4; Li and Dewey 2011).
An existing pipeline developed within the Trinity software package (Grabherr et al., 2011) was used to perform differential expression analysis with DESeq (Anders and Huber, 2010) which is available as part of the Bioconductor project (Gentleman et al., 2004).
Supplementary_files_format_and_content: Tab-delimited text file containing TPM values generated by RSEM where gene names are rows and columns represent all RNA-Seq samples generated in the study
|Feb 02, 2015
|Last update date
|May 15, 2019
|Francis Crick Institute
|Cancer Epigenetics Laboratory
|1 Midland Road
|Dynamic regulation of the histone variant H1.0 contributes to intratumor epigenetic and functional heterogeneity [RNA-Seq]
|Dynamic regulation of the histone variant H1.0 contributes to intratumor epigenetic and functional heterogeneity